The present invention relates to peptides having anti-inflammatory properties. In particular, the present invention relates to peptides through actions on cells that modify the leukocyte recruitment and activation cascade and the actions of mediators released from inflammatory cells on target cells and tissues, including smooth muscle of animals, as well as humans.
Immediate or Type 1 allergic reactions are largely attributed to IgE antibodies, although IgG antibodies can participate in and modulate allergic reactions. The allergy is generally caused by the activation of a subpopulation of immune cells, the mast cells and basophils. When antigen reacts with IgE antibody receptors on the cell""s surface the chemical mediators initiate the allergic reaction by acting on adjacent immune, epithelial, endothelial and smooth muscle cells and promote, in a longer term, the influx of other inflammatory and immune cells (neutrophils, eosinophils, monocytes, lymphocytes) into tissue. This influx of inflammatory cells predisposes the patient to recurrent and sometimes delayed, or prolonged allergic or hypersensitivity reactions. A distinction between immediate and delayed allergic reactions and delayed, chronic immune injury can also be made. The Type 1 allergic reactions are defined according to the location where they occur. Asthmatic reactions occur in the lungs, rhinitis in the nose, conjunctivitis in the eyes, and atopic dermatitis in the skin, systemic allergic reactions in the circulation and intestinal reactions in the gastrointestinal system.
Asthma can be defined clinically as a condition of intermittent, reversible airways obstruction, and manifests itself as several clinical entities: allergic asthma, bronchial asthma, exercise induced asthma (EIA), chemical induced asthma, and status asthmaticus Asthma can be divided into two types. Extrinsic asthma is generally triggered by external agent such as allergens (dust mites, pollen, stings, drugs, or foods), and is commonly diagnosed in early life. Intrinsic asthma, which generally develops later in life, can be triggered by congested and inflamed tissues, infection, endogenous catecholamines (e.g. adrenaline), drugs (e.g. aspirin), stress or exertion.
Rhinitis, allergic conjunctivitis and atopic dermatitis are inflammations of the nasal mucosa, eyes and skin, respectively, often due to allergens such as pollen, dust or other airborne substances.
Anaphylactic shock, the most severe form of allergy, is a medical emergency. It is often severe and sometimes can provoke a fatal systemic reaction in a susceptible individual upon exposure to a specific antigen (as wasp venom or penicillin) after previous sensitization. Anaphylactic shock is characterized by respiratory symptoms, fainting, itching, urticaria, swelling of the throat or other mucous membranes and a sudden decline in blood pressure. Symptoms of anaphylactic shock include dizziness, loss of consciousness, laboured breathing, swelling of the tongue, blueness of the skin, bronchospasm, low blood pressure, and death.
Inflammatory response syndrome (IRS)xe2x80x94is an inflammatory response to a wide variety of clinical insults. IRS occurs as a result of extensive tissue damage and necrosis or the invasion of microorganisms, with the release of chemical mediators or cellular by-products such as the cytokines, lipid metabolites and autocoids. These mediators can be released in response to tissues and cells affected by infections, shock (endotoxemia, blood loss, blunt trauma), hypoxemia, radiation, bums, organ transplants, graft rejections. The proinflammatory IRS response is primarily responsible for the development of organ dysfunctions, such as acute lung injury, acute respiratory distress syndrome (ARDS), damage to gastrointestinal dysfunction (ileus, changes in permeability, pancreatitis like problems), and dysfunctions of the kidney, heart, liver and brain.
Amino Acids: Abbreviations, Letter Code and Linear Structure Formula.
L-amino acids are identified by capital letters (e.g. Tyr (Y)), whereas the corresponding D-isomeric form of the amino acid is identified by small letters (e.g. tyr (y)).
Over the last few decades salivary gland growth factors (such as epidermal growth factor; EGF) have gained increased recognition as playing an important role in tissue repair. Another important feature of the actions of salivary gland factors, which can be growth factors and hormones, is the regulation of oral and systemic immune and inflammatory responses (Mathison et al, 1994). It is apparent that salivary gland involvement in regulating systemic immune and inflammatory responses is under the control of the sensory, sympathetic and parasympathetic nervous systems, as well as endocrine control from steroid control and peptide hormones. One aspect of nervous system regulation of the salivary glands involves the xe2x80x9ccervical sympathetic trunk-submandibular gland (CST-SMG) axisxe2x80x9d (Mathison et al, 1994; 1993), which is a distinct neuroendocrine system that is involved in regulating inflammatory responses and systemic homeostatic mechanisms (Ramaswamy et al., 1989; Mathison et al, 1995). The release of hormones from other salivary glands (e.g. parotid, sublingual, etc.) are also under the control of the nervous system.
Perturbation of the CST-SMG axis, by either performing a cervical sympathetic denervation or by removing the submandibular glands, results in an enhanced hypotensive response to intravenously administered endotoxin (Mathison et al, 1993). This observation led us to postulate the existence of factors within the salivary glands that modulate endotoxic hypotension, and we subsequently isolated a seven amino acid peptide (sequence=Thr-Asp-Ile-Phe-Glu-Gly-Gly SEQ ID NO.1; TDIFEGG submandibular gland peptide-T (SGP-T)), which at doses as low as 1 xcexcg/kg inhibits lipopolysaccharide induced hypotension (Mathison et al, 1997b). Investigation of structure activity relationship has identified the C-terminal of SGP-T, the tripeptide FEG (Phe-Glu-Gly) and its D-isomer feG, also have significant anti-inflammatory activities (U.S. patent application Ser. No. 051/395). The biological activities of SGP-T, FEG Or feG include: a significant reduction in the severity of intermediate hypersensitivity reactions in the lung (Dery et al., 1999, 2000; Befus et al, 2000a,b,c), intestine (Mathison et al, 1997b,c; 1998), heart (Turesin et al, 2000) and the cardiovascular system (Mathison et al, 1997a, 1999b, 2000b; Davison et al, 2000), inhibition of neutrophil chemotaxis (Mathison et al, 2000a) and superoxide production (Nkemdirim et al, 1998).
Inflammation
Inflammation is a defense reaction caused by tissue damage or injury, characterized by redness, heat, swelling, and pain. Inflammation is the result of a response of the body""s defense system that localizes and normally eradicates the irritant stimulus and repairs the surrounding tissue. Inflammation is a necessary and beneficial process, and important for survival. The inflammatory response involves four major stages: 1. dilation of capillaries to increase blood flow; 2. microvascular structural changes and escape of plasma proteins from the bloodstream; 3. leukocyte and lymphocyte rolling, adhesion and transmigration through endothelium and accumulation at the site of injury; and 4. activation of biochemical processes designed to neutralize and eradicate the offensive stimulus and initiate tissue repair.
B.1. The Leukocyte and Lymphocyte Recruitment and Activation Cascade
The leukocyte (and by inference, the lymphocyte) adhesion cascade is a sequence of activation events that ends with extravasation of a leukocyte, whereby the cell exerts its effects on the inflamed site. At least five steps of the adhesion cascade are capture, rolling, slow rolling, firm adhesion and transmigration. Each of these five steps is necessary for effective white blood cell recruitment, because blocking any of them can reduce white blood cell accumulation in the tissue. At any given moment, capture, rolling, slow rolling, firm adhesion and transmigration all happen in parallel, involving different white blood cells in the same microvessels. Some anti-inflammatory agents function as blockers, suppressors, or modulators of the inflammatory response at specific points in this sequence of events which is a part of the inflammatory process. The principal clinical hallmarks of inflammation are: rubor (redness); dolor (pain); calor (heatxe2x80x94but only of skin and extremities); tumor (swelling); and functio laesa (loss of function). Redness is caused by increased blood flow to the site, due to the action of mediators, axon reflex, and local increase in the hydrogen ion concentration. Heat is also due to increased blood flow and greater local cellular metabolism.
Swelling is the result of increased blood flow, oedema, infiltration of cells, and the proliferation of connective tissue in subacute-to-chronic lesions. Pain is due to the effects of mediators on sensory nerves and the stretching of these nerves due to swelling. Loss of function is due to replacement of parenchymal tissue (e.g., damaged myocardium); reflexive disuse due to pain; and mechanical, as when a joint either swells during acute inflammation or scar tissue formation in a chronic lesion. Thus, the inflammatory response can viewed as is a dynamic, changing process and the longer the process continues the likelihood that irreversible alteration of parenchymal tissue (e.g., scarring) occurs increases.
An essential point of this application is regulation of the severity of immediate or Type 1 hypersensitivity (anaphylactic) reactions and their associated clinical conditions, the inflammatory response syndrome and other inflammations.
B.2. Selectins and Integrins: Their Role in Cell Adhesion and Activation
Adhesion receptors are involved in many leukocyte functions including haematopoiesis, migration, activation, mediator generation and apoptosis. The leukocyte adhesion receptors are involved in many biological processes such as allergic disease, other types of inflammation, wound healing, thrombogenesis, atherogenesis and embryogenesis.
The adhesion receptors have been primarily studied for their role in directing leukocyte migration through vascular endothelium, and are intimately involved in all the processes described in the leukocyte recruitment and activation cascade described above.
The leukocyte adhesion receptors comprise several large family of cell surface proteins including the selectins, integrins, immunoglobulins and their counter receptors. There are three selectins. E-selectin expressed on endothelium, P-selectin expressed by platelets and endothelium and L-selectin expressed on most leukocytes. L-selectin is constitutively expressed but shed on cellular activation as a result of the actions of a membrane bound metalloproteinase. The ligand for L-selectin on inflamed venular endothelium has not been identified, although L-selectin does bind to PSGL-1. The selectins mediate capture of leukocytes under flow conditions, and mediate the rolling of leukocytes on endothelial cells.
Integrins are receptor proteins which are of crucial importance as they facilitate and promote cell binding and responses to the extracellular matrix. Functional integrins consist of two transmembrane glycoprotein subunits that are non-covalently bound. Those subunits are called alpha and beta. The alpha subunits all have some homology to each other, as do the beta subunits. The receptors always contain one alpha chain and one beta chain and are thus called heterodimeric. To date, 16 alpha and 8 beta subunits have been identified, which combine in different ways to form 22 natural integrins. Integrins can adhere (bind) an array of ligands with common ligands being fibronectin and laminin.
Integrins are involved in a wide range of biological functions including maintenance of tissue homeostasis through binding to matrix proteins, and one of their more intensely studied functions is their involvement in leukocyte migration. The beta 2 (CD18) leukocyte integrins comprise four members CD11a-d/CD18. CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1) are expressed on all leukocytes and are involved in a range of functions including transmigration through endothelium and cell activation. Three receptors for the integrins have been identified: ICAM-1 and 2 are expressed on endothelium and ICAM-3 expressed on most leukocytes. The beta 1 integrins (eg. the CD49 series) are also intimately involved in the biological function ascribed to the beta 2 integrins.
Tissue Specific Inflammation
C.1 The Respiratory System
Bronchial asthma is usually a chronic (long-term) disease affecting the bronchial tubes (bronchi, breathing tubes or airways) of the lungs and the sympthoms of asthma are the result of constriction or narrowing of irritable bronchial tubes. This constriction is caused by bronchial tube muscle spasm and narrowing due to inflammation, most frequently provoked by antigen induced release of histamine, leukotrienes and other chemical mediators from mast cells. With intrinsic asthma these mediators are released without an allergic trigger (i.e. following a cold or with exercise). The net result of the asthmatic attack is muscle spasm, inflammation, edema (swelling), and increased mucus production within the bronchi. The process generates symptoms within 15-30 minutes of exposure (immediate response) and which generally subside within an hour. In some individuals a delayed response (late phase reaction) occurs 3-4 hours following the immediate or initial response. It should be noted, however, that the timing of these reactions exhibit marked variability between patients and can be shorter or longer in onset and duration. The late phase reaction, which probably develops from an inflammatory reaction, may last many hours or days and is frequently associated with increased bronchial hyperreactivity or irritability rendering the individual sensitive to a variety of inhaled irritants. The pulmonary inflammation that develops with asthma is due to the influx of inflammatory cells into the lungs using the leukocyte adhesion cascade described previously.
Endotoxin and other pulmonary and systemic inflammations can also cause severe and prolonged lung inflammation. In sheep receiving intravenously LPS, pulmonary dynamic compliance was reduced 30 min post-LPS (Wheeler et al., 1990). This acute change in lung response probably reflects a rapid onset of LPS actions on circulating leukocytes and the endothelial cells of the pulmonary blood vessels with a consequent release of myoactive inflammatory mediators. It is possible that the mediators effecting changes in pulmonary dynamic compliance could also contribute to the delayed influx of cells into the lungs. Our own recent studies indicate that inflammatory reaction occurs for several days after the original endotoxic insult, as reflected in enhanced lung inflammation at 24 and 48 h after LPS administration and the enduring (at least 5 days) increase in the number of circulating white blood cells (Fialho de Aranjo et al., submitted for publication).
C.2 The Cardiovascular System
Endotoxin consistently results in a decrease in ventricular compliance in several animal species including humans, dogs and pigs. In rats with their submandibular glands removed endotoxin provokes a decrease in Pmaxxe2x88x92dP/dt (a measure of ventricular compliance) (Mathison et al, 1999a). In vitro studies have shown that changes in heart function induced by LPS are reflected by in vivo alterations in myocyte function. In a variety of species, such as the guinea pig, the rabbit and the rat reduced myocardial contractility is develops in septic hearts. Endotoxemia also produces structural changes to the heart, such as enhanced stiffness of the septic myocardium due to a decrease in myocardial collagen, an increase in interstitial water and the shrinkage of the myocytes. These changes contribute to systolic and diastolic myocardial dysfunction and in particular to the reduced ventricular diastolic relaxation. The final effectors of endotoxin induced changes in heart function are unknown, but neutrophils and macrophages/monocytes do contribute to alterations in myocyte function.
Anaphylactic reactions also can cause modification of heart function and are associated with an increase in neutrophil influx into the heart tissue (Turesin et al, 2000). This inflammation of the heart is effected by the leukocyte adhesion cascade described previously.
C.3. The Intestine
A variety of inflammatory insults such as food hypersensitivities, allergies, proliferation of bacteria or the development of an excessive burden of resident bacteria can contribute to intestinal tissue injury and inflammation. For example, an intense allergic reaction or the response to ingested bacterial toxins through a variety of mechanismsxe2x80x94from release of tissue injuring enzymes to dramatic decrease in tissue blood flowxe2x80x94can produce pronounced changes in intestinal motility and cause extensive tissue damage, particularly to the intestinal mucosa. The immediate hypersensitivity response, designed to expel the offending substances through increasing intestinal motility and secretion, in certain cases produces a significant xe2x80x9ccollateral damagexe2x80x9d to adjacent healthy tissues. The maintenance of coordinated smooth muscle activity and the integrity of the mucosal barrier is essential not only for proper functioning of the intestine but also for the maintenance of overall systemic health.
Other inflammatory insults may not produce such rapid and severe damage to the intestine as an allergic or an endotoxic reaction, but since they are generally of more prolonged duration (days to years), the progressive movement of inflammatory cells (neutrophils, monocytes and lymphocytes) into the intestinal tissues produces damage to healthy tissues that can compromise repair processes. These prolonged inflammatory insults can result in the development of severe and chronic inflammation apparent in such conditions as inflammatory bowel disease (IBD). Control of inflammation thus involves a delicate balance between containment and removal of the precipitating agent and minimizing the xe2x80x9ccollateral damagexe2x80x9d while at the same time initiating tissue repair and regeneration.
The intestine is the major organ affected by shock in rodents, pigs and humans. The shock response occurs consequent to extensive pooling of blood in these organs. (Mathison et al, 1990), and associated poor blood flow to the intestine. The resultant decreased oxygen extraction (Nelson et al, 1988) along with the release of cytotoxic molecules from inflammatory cells results in extensive mucosal damage and causes the intestine to lose coordinated motility patterns or to become totally inactive (a condition know as ileus). The coordinated contractile activity of the intestinal smooth muscle, necessary for effective and efficient intestinal transit, also is disrupted (Hellstrom et al, 1997) resulting in prolonged periods of stasis. All these factors, through increased enteric bacterial proliferation and translocation (Carrico et al, 1985), can contribute to the evolution of further organ dysfunction and progression of the systemic inflammation and sometimes localized or generalized sepsis.
Eskanadari and coworkers (1997) showed that peritoneal sepsis also provoked an increase in IL6 and CD14 (the LPS receptor on macrophages) in the intestinal smooth muscle. Concomitant with these changes in cytokine and macrophage receptor profiles was a decrease in the myogenic response of jejunal circular muscle to the myotropic agent, bethanchol. The activation of macrophages by peritoneally injected LPS was associated with a rapid and transient expression on resident muscularis macrophages (apparent only at 1 hour, as revealed with LFA1 (CD11/CD18) (Eskandari et al, 1997). The peptide feG was shown to reduce the number of CD14 and CD11b positive macrophages in the muscle layer of the rat intestine (Mathison et al, 1999b, 2000b; Davison et al, 2000).
New and effective methods are required for the maintenance of organ function in all forms of toxic reactions, whether caused by hypersensitivities to antigen, viruses, bacterial endotoxins and exotoxins, or fungi. The endogenous regulators of inflammation, produced in the salivary glands, have been demonstrated to be potent anti-inflammatory agents. Although their primary role is classically considered to be the regulation of inflammatory responses in the mouth, it is now apparent that these glandular factors have effects on the whole of the gastrointestinal tract and generalized systemic effects as well (Mathison et al, 1994; 1997). Salivary gland growth factors and small peptide hormones may be used to treat a variety of inflammatory disorders.
It has been discovered that novel compositions of matter, mainly analogues of the tripeptide FEG which consist of di- and tripeptides containing substituted L-amino acids and/or their optical isomers D-amino acids, are potent modulators of the inflammatory reactions precipitated by allergic and endotoxic reactions.
The invention is directed to a peptide of the formula:
X-R.sub.1 -R.sub.2 -R.sub.3 -Yxe2x80x83xe2x80x83(I)
or
X-R.sub.1 -R.sub.2 -Yxe2x80x83xe2x80x83(II)
wherein X is selected from the group consisting of H and acetyl; R.sub.1 is selected from the group consisting of D or L-phenylalanine; tyrosine; tryptophan; phenylglycine; Nor-methylphenylalanine; cyclohexylalanine; and norleucine; R.sub.2 is selected from the group consisting of D or L-glutamate; and aspartate; and in the case of peptide (I), R.sub.3 is selected from the group consisting of glycine; D or L-alanine; beta-alanine; valine; leucine; isoleucine; sarcosine; and gamma-aminobutyric acid or another aliphatic amino acid; and Y is selected from the group consisting of OH or NH.sub.2, except the dipeptides
H-L-Phe-L-Glu-OH
H-L-Trp-L-Glu-OH
H-D-Phe-D-Glu-OH
H-D-Trp-D-Glu-OH.
In the peptide according to the invention, X can be hydrogen, R.sub.1 can be D-phenylalanine, D-tyrosine or D-tryptophan, R.sub.2 can be D-glutamate, R.sub.3 can be glycine and Y can be NH.sub.2 or OH.
The invention is also directed to a pharmaceutical composition containing a peptide according to the invention, wherein the peptide can be present in admixture with a pharmaceutically acceptable carrier. The peptide can be present in admixture with another therapeutically active agent.
A treatment schedule composed of daily or twice daily administrations of the peptides which can be administered for once or successive dosing over many days with medications that can be administered by inhalation, orally subcutaneously or intravenously, can be utilized. Appropriate pharmaceutically acceptable carriers can be used in treatment administration.
The invention is also directed to a method of modulating inflammatory reactions in an animal which comprises administering to the animal a peptide according to the invention in an amount ranging from about 0.1 to about 1000 ig/kg. and a method for the treatment of allergic disorders in an animal using pharmaceutical compositions according to the invention. The disorder can be an intestinal allergy, asthma, rhinitis, an anaphylactic reaction. The animal can be a human being.
The invention is also directed to a method for the treatment of a toxic immunological reaction using pharmaceutical compositions according to the invention. The toxic immunological reaction can involve products released or on the surface of Gram-negative bacteria, Gram-positive bacteria, fungi, viruses or parasites. The immunological toxic reaction can involve inflammation of the respiratory system, inflammation of the gastrointestinal tract, inflammation of the heart or cardiovascular system, or inflammation of the skin, eyes and kidneys.
The invention also pertains to a method of manufacturing a medicament using a peptide according to the invention, in an amount effective to produce an therapeutic response.
The peptide can be selected from the group consisting of:
peptide #1 H-D-tyr-D-glu-Gly-OH; peptide #2 H-D-trp-D-glu-Gly-OH; peptide #3 H-D-NMef-D-glu-Gly-OH; peptide #4 H-D-cha-D-glu-Gly-OH; peptide #5 H-D-nle-D-glu-Gly-OH; peptide #6 H-D-phe-D-glu-Gly-NH.sub.2; peptide #7 H-D-tyr-D-glu-Gly-NH.sub.2; peptide #8 H-D-trp-D-glu-Gly-NH.sub.2; peptide #9 H-D-NMef-D-glu-Gly-NH.sub.2; peptide #10 H-D-cha-D-glu-Gly-NH.sub.2; peptide #11 H-D-nle-D-asp-Gly-NH.sub.2; peptide #12 H-D-tyr-D-glu-OH; peptide #13 H-D-trp-D-glu-OH; peptide #14 H-D-NMef-D-glu-OH; peptide #15 H-D-cha-D-glu-OH; peptide #16 H-D-nle-D-glu-OH; peptide #17 H-D-phe-D-glu-NH.sub.2; peptide #18 H-D-tyr-D-glu-NH.sub.2; peptide #19 D-trp-D-glu-NH.sub.2; peptide #20 H-D-NMef-D-glu-NH.sub.2; peptide #21 H-D-cha-D-glu-NH.sub.2; peptide #22 H-D-nle-D-asp-NH.sub.2.